The MLL–Menin Interaction is a Therapeutic Vulnerability in NUP98-rearranged AML

Chromosomal translocations involving the NUP98 locus are among the most prevalent rearrangements in pediatric acute myeloid leukemia (AML). AML with NUP98 fusions is characterized by high expression of HOXA and MEIS1 genes and is associated with poor clinical outcome. NUP98 fusion proteins are recruited to their target genes by the mixed lineage leukemia (MLL) complex, which involves a direct interaction between MLL and Menin. Here, we show that therapeutic targeting of the Menin–MLL interaction inhibits the propagation of NUP98-rearrranged AML both ex vivo and in vivo. Treatment of primary AML cells with the Menin inhibitor revumenib (SNDX-5613) impairs proliferation and clonogenicity ex vivo in long-term coculture and drives myeloid differentiation. These phenotypic effects are associated with global gene expression changes in primary AML samples that involve the downregulation of many critical NUP98 fusion protein-target genes, such as MEIS1 and CDK6. In addition, Menin inhibition reduces the expression of both wild-type FLT3 and mutated FLT3-ITD, and in combination with FLT3 inhibitor, suppresses patient-derived NUP98-r AML cells in a synergistic manner. Revumenib treatment blocks leukemic engraftment and prevents leukemia-associated death of immunodeficient mice transplanted with NUP98::NSD1 FLT3-ITD-positive patient-derived AML cells. These results demonstrate that NUP98-rearranged AMLs are highly susceptible to inhibition of the MLL–Menin interaction and suggest the inclusion of AML patients harboring NUP98 fusions into the clinical evaluation of Menin inhibitors.

apoptotic cells, defined as the Annexin V-positive cells, was determined by flow cytometry.

Cell viability assay
Cells transduced with NUP98::NSD1 and NUP98::DDX10 fusion genes were seeded in biological triplicates and treated with revumenib (Syndax Pharmaceuticals) or DMSO, at indicated concentrations every 48 or 72 hours. Cell numbers were determined in regular intervals with the Intellicyt iQue Screener (Sartorius AG, Goettingen, Germany) and analyzed with iQue ForeCyte Software. The data was plotted with GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, California USA).

Cytospin Analysis
To assess cell morphology, cells were washed with PBS and cytocentrifuged onto glass slides. Slides were fixed in methanol for 2 min and stained using Wright-Giemsa staining or with Epredia TM Shandon TM Kwik-Diff TM staining kit (Thermo Fisher Scientific). Slides were imaged on a Leica DM6 (Leica Microsystems) bright-field microscope.

Real-time quantitative PCR
RNA was isolated using the NucleoSpin RNA purification kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. Concentration and quality of the RNA was determined with a NanoDrop 1000 (ThermoFisher Scientific). For cDNA synthesis, 500 ng of RNA were reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific) according to the manufacturer's protocol. Quantitative real-time polymerase chain reactions (Q-RT-PCR) were performed in triplicates with 1 µL cDNA using SYBR Green gene expression master mix on a CFX384 Real-Time PCR detection system (Bio-Rad Laboratories, California), 30 seconds at 95°C followed by 35 cycles at 95°C for 15 seconds and 60°C for 30 seconds. Fold change relative to the control condition was calculated by the comparative ΔΔ cycle threshold method using TBP as the housekeeping gene for normalization.

Clonogenic assay in methylcellulose
Primary patient samples were treated with 250 nM revumenib and grown in SFEMII media supplemented with cytokines as described above for 12 days. Cells were washed with PBS and plated in triplicates on methylcellulose media supplemented with human cytokines (Methocult H4435, StemCell Technologies) at a density of 105 cells. Cells were incubated at 37 °C, 5% CO2 for 14 days before colonies were counted. Colonies were quantified using a Leica DMi8 inverted microscope (Leica Microsystems).

Sanger sequencing
To confirm the presence of the NUP98::NSD1 fusion transcript and activating FLT3-ITD mutations in samples Sanger sequencing was used. Briefly, total RNA (500 ng) was extracted from patient samples using the NucleoSpin RNA purification kit (Macherey-Nagel, Düren, Germany) and reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The NUP98::NSD1 fusion transcript was amplified from the cDNA using specific primers (Table S4)

RNA sequencing:
For the in vitro RNA-seq experiment, we treated 3 different NUP98::NSD1 (FLT3-ITD) primary AML samples in 3 independent experiments either with revumenib (250 nM) or DMSO (0.1%). We used FACS to separate leukemic cells from non-leukemic cells, MSCs and debris from co-culture. RNA was isolated from CD45+, CD33+ FACS sorted AML cells using the NucleoSpin RNA purification kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. For gene expression analysis, reads were mapped to the human genome (Hg38) using STAR (v2.2.0c). Only reads that mapped to unique genomic locations (MAPQ > 10) were used for downstream analysis and differentially expressed genes between the treated groups were identified using DESeq version 2.

Western blotting
Equal numbers of pelleted cells were washed with ice-cold phosphate-buffered saline (PBS), lysed, and protein was extracted using RIPA buffer. Protein concentration was measured using the Pierce BCA Protein Assay. Samples were separated on 8% sodium dodecyl sulfate-polyacrylamide gels and proteins were transferred to nitrocellulose membranes at 120 V for 60 min in 1× transfer buffer. Membranes were blocked with 5% non-fat dry milk in TBS-T for 1 hr at room temperature and incubated with primary antibodies in TBS-T overnight at 4°C. Afterwards, the blots were washed and incubated with HRP-conjugated goat anti-mouse IgG at RT for 1 h. After washing three times for 5 min, protein bands were visualized through chemiluminescence reaction (SuperSignal West Pico, Thermo Scientific) according to the manufacturer's protocol. The intensity of the signals was measured by ImageJ software (version no.

Data quantification and statistical analysis
RNA-seq and t-SNE analysis were performed as described in the Supplemental Methods. The software GSEA 4.1.0 was used for enrichment analysis of altered biological pathways. Statistical analyses, including non-linear regression analyses, ttests, and ANOVA were performed with the Prism software package, version 8.0. Data obtained from multiple experiments were reported as mean ± SD. If not stated elsewhere, differences with p < 0.05 were recognized as statistically significant.

Supplemental tables
Supplemental Table 1 Individual characteristics of the patient samples used in this study.